Invasive foreign nucleic acids such as transposons and viruses pose a threat to all organisms, potentially causing genome instability and disease, including cancer and infertility. Fortunately, organisms have evolved a vast array of protein and nucleic acid-based mechanisms to defend against intruders. In animals, germline-expressed small RNAs known as piRNAs interact with PIWI-clade Argonaute proteins, which together promote genome stability and fertility, in part by silencing transposable elements. Yet piRNAs exhibit remarkable sequence diversity and often map to unique sequences in the genome. In the nematode worm C. elegans, for example, tens of thousands of piRNAs are individually expressed from mini-genes and lack perfectly complementary targets elsewhere in the genome. Therefore the function of piRNAs has remained mysterious. We have recently shown that piRNAs can recognize their targets via imperfect base pair interactions and trigger stable and heritable epigenetic silencing, which can be maintained by RNAi and heterochromatin factors, independently of piRNAs. The goal of this proposal is to answer several fundamental questions regarding this RNA-mediated defense system, including: How do piRNAs recognize foreign nucleic acids? What are the key molecular and genetic determinants of piRNA targeting? How does piRNAs targeting trigger an epigenetic silencing? During the K99 (mentored) phase of this fellowship, I will develop biochemical assays and genetic reporters to understand how piRNAs recognize their targets. This information will aid the development of bioinformatics tools to predict and analyze in vivo piRNA targets. In addition, I will perform a genetic screen to identify factors required for piRNA- mediated targeting and silencing. During my R00 phase, I will further use a combination of biochemical, genetic and bioinformatics approaches to study the biogenesis of piRNAs and mechanism of piRNA-induced gene silencing. The studies proposed here will provide important insights into role of piRNAs in genome surveillance and epigenetic regulation and their role in disease pathogenesis. Furthermore, I will obtain reagents, tools and skills necessary to launch my independent research program.